66 R. MARKHAM 



several diameters apart. The proportion of the material iii the form of 

 tactoids depends upon a number of factors, the principal one being the 

 electrostatic repulsion between the rods, so that if swamping amounts of 

 ions are present, as in strong salt solutions, or if the surface charge, which 

 is mainly due to carboxyl groups, is reduced by lowering the pH, the number 

 and the internal density of the tactoids increase and the "crystals" separate 

 out. 



The bottom layer is readily oriented by shear forces (Fig. 7) and it may 

 be aligned by sucking into tubes and by gradual drying; these methods have 

 been used for the preparation of much of the material used in X-ray investiga- 

 tions. It must not be thought, however, that bottom layer has unique 

 properties. The pellets of unaggregated virus obtained by ultracentrifugation 

 have very similar optical and physical properties, but because the material 

 is composed of much shorter rods, these are not manifested so readily until 

 the solutions are much more highly concentrated. 



The unaggregated virus consists of rods which appear to be of relatively 

 uniform length when examined carefully by a number of methods. It is 

 true that there is considerable objection in some quarters to the idea of the 

 complete uniformity of the length of the virus particles, but the evidence 

 for such uniformity is more or less overwhelming, or at the worst much more 

 convincing than the opposing view, which originated through the study of 

 aggregated preparations. The exact length of the particles is, however, less 

 certain, but it is of the order of 3000 A. Measurements of particle lengths of 

 this order of size present considerable practical difficulties. The most direct, 

 and in many ways the most convincing one, is by electron microscopic 

 observation. This has been brought to a high degree of refinement by Williams 

 and his associates by the use of spraying and internal standards (Williams 

 et al., 1951). Spraying is, of course, by no means without its dangers, because 

 the shear forces encountered may even break the virus rods, and the calibra- 

 tion of the internal standards also presents some problems. In addition, 

 astigmatism of the electron optics involves systematic errors which must be 

 corrected for. However, the results obtained by different workers have been 

 in very remarkable agreement. 



Another method for the direct measurement of the particle length is 

 that employed by the King's CoUege group (Wilkins et al, 1950). These 

 workers noted that the hexagonal plates found in plant cells were multiple 

 twin crystals, having the virus rods arranged in a zigzag order along a line 

 normal to the hexagon face of the hexagonal plates. The consequence of 

 this type of arrangement was that, instead of exhibiting extinction in 

 polarized hght in a plane at right angles to the hexagonal face, the crystals 

 exhibited two extinction positions at shght angles to this plane. Therefore, 

 when viewed in one extinction position the edges of the crystals appear to 



